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PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

ABSTRACT: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques.A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol.The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples.This study describes a new high-throughput TaqMan assay that very effectively detects the four Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.

SUBMITTER: Bass C

PROVIDER: S-EPMC2553798 | biostudies-other | 2008 Sep

REPOSITORIES: biostudies-other

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PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Bass Chris C Nikou Dimitra D Blagborough Andrew M AM Vontas John J Sinden Robert E RE Williamson Martin S MS Field Linda M LM

Malaria journal 20080915

<h4>Background</h4>Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared ...[more]

PMID: 18793416

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Project description:BackgroundKnockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques.MethodsFluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe - Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals).ResultsThe real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores.ConclusionThe results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput.

Project description:Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.

Project description:BACKGROUND:Mosquito-feeding assays that assess transmission of Plasmodium from man-to-mosquito typically use laboratory mosquito colonies. The microbiome and genetic background of local mosquitoes may be different and influence Plasmodium transmission efficiency. In order to interpret transmission studies to the local epidemiology, it is therefore crucial to understand the relationship between infectivity in laboratory-adapted and local mosquitoes. METHODS:We assessed infectivity of Plasmodium vivax-infected patients from Adama, Ethiopia, using laboratory-adapted (colony) and wild-caught (wild) mosquitoes raised from larval collections in paired feeding experiments. Feeding assays used 4-6 day-old female Anopheles arabiensis mosquitoes after starvation for 12 h (colony) and 18 h (wild). Oocyst development was assessed microscopically 7 days post-feeding. Wild mosquitoes were identified morphologically and confirmed by genotyping. Asexual parasites and gametocytes were quantified in donor blood by microscopy. RESULTS:In 36 paired experiments (25 P. vivax infections and 11 co-infections with P. falciparum), feeding efficiency was higher in colony (median: 62.5%; interquartile range, IQR: 47.0-79.0%) compared to wild mosquitoes (median: 27.8%; IQR: 17.0-38.0%; Z = 5.02; P < 0.001). Plasmodium vivax from infectious individuals (51.6%, 16/31) infected a median of 55.0% (IQR: 6.7-85.7%; range: 5.5-96.7%; n = 14) of the colony and 52.7% (IQR: 20.0-80.0%; range: 3.2-95.0%; n = 14) of the wild mosquitoes. A strong association (?(16) = 0.819; P < 0.001) was observed between the proportion of infected wild and colony mosquitoes. A positive association was detected between microscopically detected gametocytes and the proportion of infected colony (?(31) = 0.452; P = 0.011) and wild (?(31) = 0.386; P = 0.032) mosquitoes. CONCLUSIONS:Infectivity assessments with colony and wild mosquitoes yielded similar infection results. This finding supports the use of colony mosquitoes for assessments of the infectious reservoir for malaria in this setting whilst acknowledging the importance of mosquito factors influencing sporogonic development of Plasmodium parasites.

Project description:BACKGROUND:Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS:Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS:Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.

Dataset Information

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Publications

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

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